Cytotechnology 3: 295-299, 1990. 9 1990 Kluwer Academic Publishers. Printed in the Netherlands. Technical report

Industrial production of monoclonal antibodies and therapeutic proteins by dialysis fermentation

Michael J. Comer, Michael J. Keams, Jiirgen Wahl, Michael Munster, Thomas Lorenz, Berthold Szperalski, Stefan Koch, Ulrich Behrendt and Herwig Brunner Boehringer M a n n h e i m GmbH, Research Centre Penzberg, Nonnenwald, 8122 Penzberg, F R G Received 14 August 1989; accepted in revised form 9 November 1989

Key words: large-scale cultivation, monoclonal antibodies, therapeutic proteins

Abstract A novel and powerful fermentation method is reported for the large-scale growth of mammalian cells and their secreted products. The system described illustrates many of the advantages of conventional batch fermentation processes but in addition has been shown to yield cell densities in excess of 1 • 107 cells/ml with concomitant increase in product concentration. Abbreviations: MAb - Monoclonal Antibody, STR - Stirred Tank Reactor, FBS - Foetal Bovine Serum

Introduction Since the introduction of hybridoma technology by K6hler and Milstein (1975) the demand for monoclonal antibodies has witnessed a rapid increase. In vitro production of MAbs is now a prerequisite for many further applications and technologies. Further, genetic engineering techniques have enabled high level expression of numerous therapeutic proteins e.g. tissue plasminogen activators (t-PA), erythropoietin (EPO) and monoclonal antibodies. The possibility to express natural molecules in in vitro propagation makes them attractive as potential pharmaceutical products. Many methods are currently available for large scale cultivation of mammalian cells, for example cultivation in deep tank culture vessels with the refinement of microencapsulation (Lim and Sun, 1980), microcarriers (van

Wezel, 1967), spin-filter (Tolbert et al., 1981) or entrapped or supported as in hollow fibres (Knazek et al., 1972; Schonberg and Belfort, 1987), ceramic matrices (Berg and B6deker, 1988) etc. These methods, in general, all strive to achieve an increased cell density above that normally obtained in culture flasks with a predicted increase in product concentration. However, it has been observed that cell density in batch culture is generally limited by nutrient exhaustion and the accumulation of waste products (Birch and Cartwright, 1982; Reuveny et al., 1986). To alleviate these problems perfusion and continuous fermentation techniques have been developed and successfully employed to achieve cell densities in the region of 1 • 107 cells/ml. However, for industrial purposes such systems are not always desirable since in particular the defining of batches or lots of the therapeutic proteins re-

296 Table 1. Advantages of stirred tank reactor technology

1. 2. 3. 4. 5. 6. 7.

Flexibility of plant. Simple scale-up potential. No special construction problems. Limitedspecialized knowledge required for operators. Systemsare considered to have 'long-life' potential. Multiple operation modes (batch, fed-batch, continuous with/without biomass feedback). Simpleand proven monitoring systems/technologyavailable.

quired is more complex than the established batch processes. In this technical note we describe a novel and powerful m e t h o d for the production of complex biomolecules from mammalian cells on an industrial scale. This system demonstrates many of the advantages o f a conventional batch fermentation process but realises the higher cell densities with the concomitant increase in product concentration (Kearns et al., 1985, 1986).

C o n c e p t i o n o f the s y s t e m

In the development of the described system we decided at an early stage to utilize stirred tank reactor technology (STR) as our baseline. The philosophy behind this decision lay with our experience in the field of large-scale microbial fermentation and the proven capability o f STR technology (Table 1). Naturally, STR technology has its limitations. The fermentation runs are normally processes where m e d i u m containing 10% foetal bovine serum yields cell densities in the order o f 1 - 2 • 106 cells/ml with M A b concentrations (e.g. mouse-mouse hybridoma) ranging between 1 5 30 mg/l. The termination of growth may be attributed to exhaustion of nutrients and/or the accumulation of toxic components in the medium. Furthermore, the ratio of M A b protein to total protein concentration in the culture supernatant can be of paramount significance for downstream processes. In a normal batch fermentation, using 10% FBS, the ratio o f product to undesired proteins is often less than 0.5%. This ratio can certainly be improved by the use of serum-free

medium. However, low product yields must frequently be accepted for this substition. Our aims in the development of the reactor system described were to increase or improve; - the cell densities above those normally obtained in STRs, - the life-span of our cultures, - the ratio of secreted product to total protein in the supematant, - final product concentration. This we believed could be accomplished by controlling nutrient supply and waste removal in a modified batch fermentation in order to achieve high cell densities since our observations indicated that product formation was dependent on cell metabolism during the proliferation phase.

D e s c r i p t i o n o f the s y s t e m a n d results

Figure 1 shows a diagram of the culture system. In preliminary experiments a modified hollowfibre blood dialysis module was built into a fermenter designed for the cultivation of mammalian cells. These modules were subsequently replaced by 'tailor-made' modules with varying properties e.g. the construction material, the surface area (0.01 m2fi - 0.3 m2/l), and the molecular weight cut-offs between 10,000 and 100,000 Daltons, and which were suitable for the production of specific biomolecules. Different membranes from different suppliers were used and their suitability was largely dependent on the cell line and the desired product. Examples of the manufacturers o f such membranes are: Amicon, Akzo, Millpore, Asahi, Medical, Romicon, B e r g h o f etc.

297

~

20 4 1

15

10 I E --1

":," :.'.

:':.

/:'----2 ~

Nm

I

II

III

IV

V

clone

J

Fig. 2. Process improvementthrough dialysis fermentation:

Fig. 1. A diagrammatic representationof the dialysis fermenter:

I) Fermenter vessel 3) Dialysis module 4) Pump 2) Medium 2) Reservoir.

mouse-mouse hybridomas. cell density. M MAb concentration. 40

30

Basal m e d i u m excluding FBS is p u m p e d through the module which in turn acts to r e m o v e low molecular weight waste products whilst replenishing essential nutrients consumed by the cells. This external m e d i u m reservoir m a y be operated as a closed or open system depending on the metabolism o f the cell line being cultivated. The size o f the reservoir is dependent on the cell line, the cultivation time, the m e d i u m and the product. The culture contained within the fermenter can be run with serum supplemented, serum depleted or serum-free media. Using this system cell densities in excess o f 1 x 107 cells/ml with viabilities greater than 90% have been obtained and e.g. in the case o f human-human hybridomas, with an increase in product concentration o f between 15 and 30 fold. Figures 2 and 3 show the results achieved by this kind of dialysis fermentation in contrast to batch for various h u m a n and murine hybridomas. The data shows that substantial increases in cell numbers and M A b concentration were obtained in every case in comparison to normal batch. The increase in product concentration ratios were to have significant effects on the yields after downstream processing. Similar data have been obtained for the secretion o f therapeutic proteins by the cultivation o f various genetically modified chinese hamster ovary clones.

v .~ 2o

II

III

clone

Fig. 3. Process improvementthrough dialysis fermentation:

human-human hybridomas. ceil density. N MAb concentration,za

Figures 4 to 6 show the results of a dialysis fermentation run of a mouse-mouse hybridoma secreting IgG which under normal batch conditions with a corresponding cell density of 1 x 106 cells/ml produces 4 0 - 5 0 mg/l MAb. By employing dialysis technology this particular process could be improved approximately 10 fold. W e have attributed this to the improved supply o f nutrients and the removal o f waste products (Fig. 5) through the dialysis module integrated in the vessel.

Conclusion This technology has enabled us to produce quantities o f monoclonal antibodies and therapeutic

298 100

o o"

o.

...

75

~ so E

25

@

o

0

,

i

o

i

so

~oo

~so

F e r m e n t a t i o n Time

2oo

2~o

(h)

Fig. 4. Dialysis fermentation of a mouse-mouse hybridoma:

growth kinetics. . . . total cell number,

viable cell number.

A f u r t h e r i m p o r t a n t f a c t o r o f the s y s t e m is its ' b a t c h f e r m e n t a t i o n ' c h a r a c t e r i s t i c s , w h i c h is p r e f e r a b l e in t h e d e f i n i t i o n o f p r o d u c t lots o r b a t c h e s , a n d is i m p o r t a n t in t h e p r o d u c t i o n o f p r o t e i n s intended for therapeutic application. I n c o n c l u s i o n , it c a n b e s a i d t h a t this d i a l y s i s f e r m e n t a t i o n s y s t e m h a s p r o v e d to b e a n e x t r e m e l y v a l u a b l e a n d p o w e r f u l t o o l in the p r o d u c t i o n o f biomolecules from mammalian cells and demonstrated many of the advantages of both batch and c o n t i n u o u s f e r m e n t a t i o n p r o c e s s e s in a s i n g l e system.

500

g=g m =.

Acknowledgements 40o

W e s h o u l d l i k e to a c k n o w l e d g e t h e t e c h n i c a l assistance of Messrs Blasey, Hildebrand and Steegrnans. This work was partially supported by the Bundesministerium for Forschung und Technologie.

300

e

200 x~

I00

0

50

100

150

250

200

F e r m e n t a t i o n Time (h)

Fig. 5. Dialysis fermentation of a mouse-mouse hybridoma:

References

production of antibody (IgG).

#

, 0

50

, ~.... : ' . ' " J , 100

........ ;", ...... i'

150

200

250

F e r m e n t a t i o n Time (h)

Fig. 6.

D i a l y s i s f e r m e n t a t i o n o f a mouse-mouse h y b r i d o m a :

substrata u t i l i z a t i o n and lactate accumulation. - - - glucose, - lactate . . . . . glutaminc.

proteins on an industrial scale up to 1000 1. Furthermore, the s y s t e m m a y be scaled up f r o m the laboratory to a production dimension with a good correlation and reproducibility. The system has also p r o v e d to be suitable for the cultivation o f cell lines o f different origin, e.g. anchoragedependent cells p r o p a g a t e d on microcarriers.

1. Berg GJ and BOdeker BGD (1988) Employing a ceramic matrix for the immobilization of mammalian cells in culture. In: Spier RE and Griffiths JB (eds.) Animal Cell Biotechnology, Vol. 3, pp. 321-335. Academic Press, London. 2. Birch JR and Cartwright T (1982) Environmental factors influencing the growth of animal cells in culture. J. Chem. Tech. Biotechnol. 32: 313-317. 3. Kearns MJ, Comer MJ, Steegmans U and Jungfer H (1985) Boehringer Mannheim GmbH, German patent: DE 3541738. 4. Keams MJ, Comer MJ, Steegmans U and Jungfer H (1986) Boehringer Mannheim GmbH, European patent: EP 0224800A2. 5. Knazek RA, Gullino PM, Kohler PO and Dedrick RL (1972) Cell culture on artificial capillaries: an approach to tissue growth in vitro. Science 178: 65-66. 6. K6hler G and Milstein C (1975) Continuous culture of fused cells secreting antibody of predefined specificity. Nature 256: 495-597. 7. Lim F and Sun AM (1980) Microencapsulated islets as bioartificial endocrine pancreas. Science 210: 908-910. 8. Reuveny S, Velez D, Macmillan JD and Miller L (1986) Factors affecting cell growth and monoclonal antibody production in stirred reactors. J. Immune. Methods 86: 53-59.

299 9. Schonberg J and Belfort G (1987) Enhanced nutrient transport in hollow-fibre perfusion bioreactors a theoretical analysis. Biotechnol. Progress 3: 80-89. 10. Tolbert WR, Feder J and Kimes RC (1981) Large-scale rotating filter perfusion system for high density growth of mammalian suspension cells. In Vitro 17: 885-890.

11. Van Wezel AL (1967) Growth of cell strains and primary cells on microcarriers in homogeneous culture. Nature 216: 64-65. Address for offprints: M.J. Comer, Boehringer Mannheim GmbH, Research Centre Penzberg, Nonnenwald, 8122 Penzberg, FRG

Industrial production of monoclonal antibodies and therapeutic proteins by dialysis fermentation.

A novel and powerful fermentation method is reported for the large-scale growth of mammalian cells and their secreted products. The system described i...
288KB Sizes 0 Downloads 0 Views